The present study aimed to investigate the protective effect of Clerodendrum infortunatum (CI) by targeting its antioxidant properties against liver damage induced by arsenic in rats. Total 18 rats weighting 140-200 g, were divided into three groups. Groups representing control and treatment groups [treated with sodium arsenite alone (3 mg/kg b.w. of rats) and in combination with methanolic extract of CI leaves (100 mg/kg b. w.)]; groups named I, II and III respectively. Sodium arsenite and CI extracts were administered orally, once daily over a period of 28 days. Body wt. of each rats were recorded on day 0, 14 and 28 days of the experiment.
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 1983-1991 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number (2017) pp 1983-1991 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.605.222 Ameliorative Potential of Methanolic Extract of Clerodendrum infortunatum Linn in Arsenic Induced Sub-Acute Hepatotoxicity in Albino Rats Pinki Singh1*, Raju Prasad2 and Shamshun Nehar3 PG Department of Zoology, Ranchi University, Ranchi, Jharkhand, India Department of Pharmacology and Toxicology, Ranchi Veterinary College, BAU, Ranchi, Jharkhand, India PG Department of Zoology, Ranchi University, Ranchi, Jharkhand, India *Corresponding author ABSTRACT Keywords Ameliorative, Clerodendrum infortunatum, hepatotoxicity, rats, Sodium arsenite Article Info Accepted: 19 April 2017 Available Online: 10 May 2017 The present study aimed to investigate the protective effect of Clerodendrum infortunatum (CI) by targeting its antioxidant properties against liver damage induced by arsenic in rats Total 18 rats weighting 140-200 g, were divided into three groups Groups representing control and treatment groups [treated with sodium arsenite alone (3 mg/kg b.w of rats) and in combination with methanolic extract of CI leaves (100 mg/kg b w.)]; groups named I, II and III respectively Sodium arsenite and CI extracts were administered orally, once daily over a period of 28 days Body wt of each rats were recorded on day 0, 14 and 28 days of the experiment At the end of treatment period, liver weight were recorded and their samples were collected for the examination of oxidative stress parameters like superoxide dismutase (SOD) and malondialdehyde (MDA) levels, and liver functioning enzyme marker i.e ALT and AST Group I showed the highest increase in final b.wt followed by Group III and then Group II Significant reduction in liver wt and SOD levels, and increment in MDA levels were observed in arsenic induced rats Increase in ALT and AST were observed on arsenic exposure Our findings suggest that CI may impede the oxidative stress, lowers the liver toxicity enzyme marker level and protect the cells which have been damaged by arsenic toxicity CI plays a protective role against arsenic-induced toxicity in liver and may potentially be used as a remedial agent Introduction Arsenic is a metalloid and widespread environmental toxicant It is potent environmental toxic agent leading to various harmful effects on the human health People get exposed to arsenic during professional and environmental processes (Ramanathan et al., 2003) Epidemiological studies of human has demonstrated direct relationship between exposure of arsenic compound in drinking water and several types of cancer (Droge, 2002) The accumulation of arsenic generates oxidative stress in the body causing fatal effects to important biological processes leading to cell death Arsenic toxicity generates reactive oxygen species (ROS) such as hydroxyl radicals (OH), superoxide anions (O-2) through chain reactions and causing oxidative damage to lipids, proteins and DNA 1983 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 1983-1991 of the body (Sarkar et al., 2003) Arsenic induced apoptotic events are triggered by increasing oxidative stress and depletion of endogenous anti-oxidant system (Droge, 2002).Therefore, the anti-oxidant enzyme activity inhibits the antioxidant/oxidant imbalance Group III (Arsenic+CI extract, n=6): Sodium arsenite @ mg/kg in combination with CI @ 100mg/kg via stomach tube for 28 days Detoxification of toxic xenobiotics or foreign compounds occurs mostly in the hepatic tissue and to a lesser extent in some extra hepatic tissues Several studies demonstrated that arsenic toxicity leads to liver injury and the extent of injury to the hepatocytes is generally detected by the activity of ALT, AST, ALP and protein etc in serum Plant Collection and preparation of extract Clerodendrum infortunatum Linn., family Verbenacae is a small shrub plant found throughout the plains of India; and in Jharkhand, is found in all districts along the streams, edges of glades and in shady places This plant has been used in Indian folks medicine; leaves, roots and flowers are used in various diseases and disorders (Dymock, 1976) Therefore, the present study was aimed to investigate ameliorative efficacy of the 80% methanolic extracts of CI against sodium arsenite induced sub-acute hepatotoxicity in albino rats The liver tissue samples were collected for the study at the end of experimental period Clerodendrum infortunatum leaves were collected just after the rainy season from the herbal garden of Ranchi Veterinary College, kanke, Ranchi The leaves were washed and shade dried for about four weeks, leaves were then pulverized and grinded to powder in mixer grinder Extraction of 80% methanol extract of powdered material of CI was done as per the standard method (Panigrahi et al., 2016) The extracts were reconstituted by dissolving the solid mass with double distilled water for the oral administration in rats Body weight and liver weight Rats of each group were weighed on 0, 14 and 28 day up to 28 days and the body weights of individual rats in different groups were recorded Liver was collected at the end of the experiment and their weight was properly recorded Materials and Methods Assessment of SOD enzyme activity Study groups In our study, 18 albino male rats with identical biological and physiological characteristics housed under standard laboratory conditions (22±2 0C, 12 h light /12 h dark cycle ) were divided into three groups Group I (control, n= 6): Normal feed and water was provided for 28 days Group II (Arsenic induced, n=6): Sodium arsenite @ mg/kg via stomach tube for 28 days The superoxide dismutase (SOD) activity was assessed according to the method described by Madesh and Balasubramanium (1998) It involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazollium dye MTT [3 – (4 – dimethyl thiazol 2-xl) 2.5 diphenyl tetrazolium bromide] to its formazan, measured at 570 nm The reaction was terminated by the addition of dimethyl sulfoxide (DMSO), which helps to solublize the formazan formed 1984 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 1983-1991 The enzyme activity has been expressed as µM MTT formazan formed / min/mg protein, using a molar extinction coefficient of 17000 M-1 cm-1.The colour evolved is stable for many hours, and expressed as µM of MTT formazan formed/mg protein or U/gm tissue Estimation of MDA level Lipid peroxidation in liver tissue homogenate was estimated in terms of malondialdehyde (MDA) production by the modified method of (Stock and Dormandy, 1971) as described by (Jain, 1988) 0.5 ml of tissue homogenate was suspended in 0.5 ml of PBS To this 0.5 ml of 30 % TCA was added Tubes were vortexed and allowed to stand on ice for at least h Tubes were centrifuged at 2000 rpm for 15 minutes ml of the supernatant was transferred into another tube and to this 0.075 ml of 0.1M EDTA and 0.25 ml of 0.67% thiobarbituric acid in 0.05M NaOH was added It was mixed and the tubes were kept in a boiling water bath for 15 and then allowed to cool at room temperature Absorbance was read at 532nm The amount of lipid peroxidation is expressed in nanomoles of MDA formed per mg of protein Assessment of Liver toxicity enzyme Test The biochemical estimations were done by using Erba Semi-autoanalyzer ALT and AST in blood was measured by modified IFCC method given by (Sannigrahi et al., 2009) ALT and AST levels were expressed as IU/L Statistical Analysis The all experimental data were expressed as mean ± standard error of mean (SEM) Results are expressed as Mean ± SEM with n equal to number of animals Data were analyzed applying one way anova using the GraphPad Prism v4.03 software program (San Diego, CA, USA), and the differences were considered statistically significant at * P